Retinal ganglion cells (RGCs) are the sole projection neurons of the vertebrate eye, and their axons comprise the optic nerve. Defects involving the optic nerve are a significant cause of blindness in childhood and RGCs are the common final target of glaucoma pathogenesis. RGCs are the first neurons born in the optic cup, from multipotent progenitor cells which later give rise to amacrines, cone and rod photoreceptors, horizontal and bipolar cells, and Muller glia. The mechanisms underlying retinal histogenesis are poorly understood, but are thought to involve intrinsic and extrinsic factors. Recently, we identified a mouse basic helix-loop-helix (bHLH) transcription factor, Math5, that is homologous to the Drosophila atonal and whose specific expression pattern in the retina is correlated with birth of RGCs (Brown et al. 1998). We have created Math5 knockout mice. The homozygotes are viable but lack RGCs and optic nerves (Brown et al 2001a). They have a concomitant increase in cone photoreceptors, consistent with a cell-fate shift. Their eyes also lack the central artery and vein, and exhibit a neovascularization phenotype with some laminar alterations, but otherwise appear normal This deletion of a single retinal neuron class is a unique finding, and significant important aspects of the adult and embryonic phenotype remain unexplored. Our preliminary data and evolutionary comparisons suggest that Math5 is downstream of Hes1 and upstream of Brn3b (Pou4f2) the transcription factor in a hierarchy of retinogenesis. Math5 -/- eyes are expected to lack all mRNAs that are uniquely required for RGC determination, differentiation and maintenance. We have characterized the human ATH5 ortholog (Brown et al. 2001b) and collected samples from patients with optic nerve aplasia, hypoplasia and glaucoma for mutation screening. Apart from Math5, three loci are known to control RGC development or number in mice-Brn3b, the Nnc1 QTL, and the classical spontaneous mutation Bst (belly spot and tail). Bst/+ mice have a similar but less marked reduction in RGCs compared to Math5 -/- mice and a comparable retinal neovascularization phenotype. Using an intersubspecific backcross, we have mapped Bst to a-1 cM interval on mouse chromosome 16, close to Hes1 and bridging two YAC contigs. Finally, using the lacZ knock-in allele we have identified a late Math5 expression domain in the hindbrain (trapezoid body) and cerebellum. These findings, and the observation of a small number of ataxic Math5 mutant mice in the N2F2 generation, suggest that Math5 may also have a secondary role in auditory processing and coordination. In this proposal, we aim: (1) to characterize the adult Math5 eye phenotype in detail, including histological analysis with a panel of retinal neuron and vascular markers, ERG and circadian rhythm physiological studies, and quantitative analysis of RGCs in Math5-/+ heterozygotes; (2) to investigate the embryological basis for the Math5 -/- phenotype, by exploring the attenuated optic stalk development and the interdependence between neuronal, vascular, and astrocyte development in Math5 -/- retinas, by using BrdU birth-dating methods to directly test the RGC-to-cone cell-fate switch model, and by generating Math5-Cre transgenic mice to fully define the lineage of Math5-positive precursor cells and test the roles of specific signaling pathways (e.g. Notch-delta) in RGC development; (3) to define target genes for Math5 and the transcriptome of developing and adult RGCs using I-gene cDNA microarrays (Farjo et al. 2001) and subtractive PCR techniques; (4) to test the role of ATH5 and POU4F2 mutations in human optic nerve a/hypoplasia and glaucoma; (5) to finely map and clone Bst, and order Bst relative to Math5 in a developmental hierarchy; and (6) to characterize the hindbrain and cerebellar Math5 expression domains, and test the role of Math5 in auditory processing and motor control.